linoleic-acid and 5-5-dimethyl-1-pyrroline-1-oxide

Lipid derived free radical in soymilks were studied by combining 5,5-dimethyl-pyrroline-l-oxide (DMPO) spin trap, chloroform-methanol extraction and electron spin resonance (ESR) spectroscopy. Five lipid derived free radical adducts: DMPO-X, DMPO-L, DMPO-R, DMPO-LOO, and DMPO-RO were presented in soymilks. The total amounts of spins increased as the soaking temperature increased from 4 °C to 50 °C and the soaking pH increased from 3 to 9 and in paralleled with the diffusion of soybean exudates to soaking water. Prolonged soaking of soybean at 50 °C resulted in a higher signal intensity of DMPO-R than that of DMPO-LOO. Soybean lipoxygenases (LOXs) were responsible for the formation of lipid derived free radicals in soymilks. Soybean exudates affected the total amounts of lipid radicals in linoleic acid (LA) - LOX model system. The relative signal intensities of DMPO-R and DMPO-LOO were depended on the contents of soybean exudates in the system.

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Glycine max; Hydrogen-Ion Concentration; Linoleic Acid; Lipids; Soy Milk; Spin Labels; Temperature

Polyunsaturated fatty acid (PUFA) peroxyl radicals play a crucial role in lipid oxidation. ESR spectroscopy with the spin-trapping technique is one of the most direct methods for radical detection. There are many reports of the detection of PUFA peroxyl radical adducts; however, it has recently been reported that attempted spin trapping of organic peroxyl radicals at room temperature formed only alkoxyl radical adducts in detectable amounts. Therefore, we have reinvestigated spin trapping of the linoleic, arachidonic, and linolenic acid-derived PUFA peroxyl radicals. The slow-flow technique allowed us to obtain well-resolved ESR spectra of PUFA-derived radical adducts in a mixture of soybean lipoxygenase, PUFA, and the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). However, interpretation of the ESR spectra was complicated by the overlapping of the PUFA-derived alkoxyl radical adduct spectra. In order to understand these spectra, PUFA-derived alkoxyl radical adducts were modeled by various alkoxyl radical adducts. For the first time, we synthesized a wide range of DMPO adducts with primary and secondary alkoxyl radicals. It was found that many ESR spectra previously assigned as DMPO/peroxyl radical adducts based on their close similarity to the ESR spectrum of the DMPO/superoxide radical adduct, in conjunction with their insensitivity to superoxide dismutase, are indeed alkoxyl radical adducts. We have reassigned the PUFA alkylperoxyl radical adducts to their corresponding alkoxyl radical adducts. Using hyperfine coupling constants of model DMPO/alkoxyl radical adducts, the computer simulation of DMPO/PUFA alkoxyl radical adducts was performed. It was found that the trapped, oxygen-centered PUFA-derived radical is a secondary, chiral alkoxyl radical. The presence of a chiral carbon atom leads to the formation of two diastereomers of the DMPO/PUFA alkoxyl radical adduct. Therefore, attempted spin trapping of the PUFA peroxyl radical by DMPO at room temperature leads to the formation of the PUFA alkoxyl radical adduct.

Topics: Alcohols; alpha-Linolenic Acid; Arachidonic Acid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Fatty Acids, Unsaturated; Free Radicals; Glycine max; Hydrogen Peroxide; Iron; Linoleic Acid; Lipoxygenase; Molecular Structure; Peroxides; Spin Trapping; Stereoisomerism

We have used the spin trap 5,5-dimethyl-pyrroline-1-oxide (DMPO) and EPR to detect lipid-derived radicals (Ld*) during peroxidation of polyunsaturated fatty acids (PUFA), low-density lipoprotein (LDL), and cells (K-562 and MCF-7). All oxygen-centered radical adducts of DMPO from our oxidizable targets have short lifetimes (<20 min). We hypothesized that the short lifetimes of these spin adducts are due in part to their reaction with radicals formed during lipid peroxidation. We proposed that stopping the lipid peroxidation processes by separating oxidation-mediator from oxidation-substrate with an appropriate extraction would stabilize the spin adducts. To test this hypothesis we used ethyl acetate to extract the lipid-derived radical adducts of DMPO (DMPO/Ld*) from an oxidizing docosahexaenioc acid (DHA) solution; Folch extraction was used for LDL and cell experiments. The lifetimes of DMPO spin adducts post-extraction are much longer (>10 h) than the spin adducts detected without extraction. In iron-mediated DHA oxidation we observed three DMPO adducts in the aqueous phase and two in the organic phase. The aqueous phase contains DMPO/HO* aN approximately aH approximately 14.8 G) and two carbon-centered radical adducts (aN1 approximately 15.8 G, aH1 approximately 22.6 G; aN2 approximately 15.2 G, aH2 approximately 18.9 G). The organic phase contains two long-chain lipid radical adducts (aN approximately 13.5 G, aH approximately 10.2 G; and aN approximately 12.8 G; aH approximately 6.85 G, 1.9 G). We conclude that extraction significantly increases the lifetimes of the spin adducts, allowing detection of a variety of lipid-derived radicals by EPR.

Topics: Acetates; Cyclic N-Oxides; Dihematoporphyrin Ether; Docosahexaenoic Acids; Electron Spin Resonance Spectroscopy; Fatty Acids, Unsaturated; Free Radicals; Half-Life; Humans; K562 Cells; Light; Linoleic Acid; Lipid Metabolism; Lipid Peroxidation; Lipid Peroxides; Lipoproteins, LDL; Molecular Structure; Spectrophotometry, Ultraviolet; Tumor Cells, Cultured